1. Physiology of Coagulation
Hemostasis is the mechanism by means of which living beings respond to a hemorrhage and involves the participation of two processes that become functional immediately after a lesion and remain active for a long period of time. The first of them is known as primary hemostasis and is characterized by the occurrence of vasoconstriction at the vascular lesion site and platelet aggregate formation. The second one is known as secondary hemostasis, being the phase in which the fibrin clot is formed due to the action of the different coagulation cascade proteolytic enzymes.
Platelet aggregate plays a key role in hemostasis in capillaries, being particularly relevant in mucocutaneous hemorrhages. In contrast, fibrin clot formation is much more important in large vessel hemostasis, being more relevant in internal hemorrhages (gastrointestinal, cerebral, etc.). The following phases can be distinguished during platelet aggregate formation: (i) platelet adhesion to the sub-endothelium surface exposed by the lesion; (ii) release of the granular content of platelets as a response to their activation; (iii) platelet aggregation with the subsequent sequestering and concentration of more platelets at the lesion site; and (iv) binding of fibrinogen as well as other coagulation proteins to the platelet surface to produce thrombin and form the fibrin clot that will allow the plates to become fused and consolidated, thus stabilizing the hemostasic clot. Furthermore, it is well known that platelet count is critical for fibrin clot formation; platelet counts below 20,000 per μl are accompanied with severe bleeding episodes.
Several cofactors and proteolytic enzymes participate in the second phase of the blood coagulation process, all referred to as coagulation factors, and it consists of several phases ending with fibrin formation from fibrinogen hydrolysis due to the action of thrombin. Furthermore the thrombin production enhances the platelet aggregate by increasing the activation and aggregation of more platelets. Thrombin is previously formed by proteolytic hydrolysis of an apoenzyme, prothrombin. This proteolysis is carried out by the serine protease FXa, which binds to the surface of the activated platelets and only in the presence of its cofactor, activated coagulation factor V (FVa), and calcium ions, this serine protease is able to hydrolyze prothrombin. FXa can occur by two separate pathways, the intrinsic pathway and the extrinsic pathway.
The intrinsic pathway consists of a series of reactions involving mainly coagulation factor VIII (FVIII), coagulation factor IX (FIX) and coagulation factor XI (FXI), in which each proenzyme is hydrolyzed, yielding its active protease form (FVIIIa, FIXa and FXIa). In each step, the recently formed proteolytic enzyme will catalyze activation of the following proenzyme to successively yield the active form. Activation of the different coagulation factors involved in the intrinsic pathway takes place; therefore, in the manner of a cascade, a deficiency of any of the proteins of the intrinsic pathway blocks activation of the following step, preventing clot formation and increasing hemorrhagic tendency. Deficiencies of different coagulation factors, for example, FVIII, FIX or FXI, cause severe hemorrhagic syndromes, such as hemophilia A, B and C, respectively.
In the blood coagulation extrinsic pathway, the TF exposed on adventitia cells at the lesion site, binds to circulating coagulation factor VII/activated coagulation factor VII (FVII/FVIIa) to form the TF::FVIIa complex and, in the presence of calcium, to act as a substrate for FX activation. The extrinsic pathway is currently considered the most relevant pathway in blood coagulation, and it is accepted that in the event of a hemorrhage produced by a vascular lesion, coagulation is triggered due to extrinsic pathway activation involving the interaction of TF with its ligand, FVII/FVIIa.
Another role assigned to the TF::FVIIa complex in coagulation is to act as a substrate so that FX activation takes place due to FVIIa As a result, basal FXa levels (<150 pM), which initially are insufficient to generate fibrin clot formation, increase. This increases in basal FXa concentrations in the presence of its cofactor, FVa, and of a cellular procoagulant surface, would be able to produce the thrombin required for fibrin clot formation. It is currently accepted that once the platelets are activated, they play a key role in blood coagulation. They provide the procoagulant surface rich in anionic phospholipids and on the other hand they expose the FVa and FXa factors stored within them. All this allows correct assembly of the different agents involved in coagulation on the surface of their plasma membranes forming the well known prothrombinase complex (which includes FXa, FVa, prothrombin, and an anionic procoagulant platelet phospholipid surface).
The theory of extrinsic pathway activation is capable to explain how coagulation begins through the role that has been attributed to the TF::FVIIa complex. One example illustrating the biological relevance of the TF::FVIIa complex in the blood coagulation process is the Disseminated Intravascular Coagulation (DIC) Syndrome. This clinical condition is associated with the intravascular release of TF and can occur in the course of severe clinical conditions (shock, sepsis, cardiac arrest, major trauma, liver disease, major surgery, burns, etc).
Evidently in a context in which FVIIa is not present, as occurs in a congenital deficiency of this factor, coagulation hypothetically will never take place since FX will not be activated at sufficient levels and, consequently, hemorrhagic manifestations should be fatal. Murine models confirm this theory and FVII deficiency is incompatible with life, being accompanied by severe fatal hemorrhages. However, congenital FVII deficiencies described in humans until now, are not always accompanied by hemorrhages. Cases of complete FVII deficiency with no clinical symptoms and occurring in healthy individuals with no hemorrhagic complications have been reported. All this suggests that other trigger coagulation mechanisms independent of FVII must exist in humans.
TF is an integral membrane glycoprotein belonging to the super-family of class II cytokine receptors specifically bonding to FVI/FVIIa and plays a relevant role in the blood coagulation extrinsic pathway. The physiological roles assigned to TF are well known; on one hand, it is a receptor specific for FVIIa and, once the TF::FVIIa complex has been formed, it acts as a substrate so that FX activation takes place. In fact, after a vascular lesion, TF, which is normally sequestered on the surface of adventitia cells externally surrounding blood vessels, comes into contact and interacts with its ligand, FVII present in blood, to form the TF::FVII complex. Once this complex is formed, FVII autoactivation takes place, yielding its active form FVIIa. There is currently extensive information on the TF::FVII complex structure. The main FVII binding sites participating in the interaction with TF are located in the first domain similar to that of the epidermal growth factor (EGF) and in the protease domain. On the other hand, it has also been reported that other less relevant binding sites participate (4-carboxyglutamate-rich domain (Gla domain) and the second EGF domain). The binding sites present in TF are located in the two type III fibronectin domains and in the intermediate region between both domains.
Recent studies have allowed identifying that TF Lysine 165 and Lysine 166 residues interact with the Gla domain of FX, both in the activated and non-activated forms. However, in contrast with that which occurs with information referring to the TF::FVIIa complex little is known about the interaction of TF with FX and FXa. First, data suggests that Lys (165 and 166) residues act as a substrate for FX activation. On the other hand, it has been recently described that TF can acts as FXa cofactor for FVII activation. That is, the binding of FVII to TF stimulates FVIIa autoactivation and FX activation. After, FXa bound to TF stimulates FVII activation which, in turn, will increase FX activation, and consequently proturombin hydrolysis and fibrin clot formation.
The inventors have discovered that the postulated role of TF as cofactor for FXa is much more relevant becoming critical for hemostasis. Despite to the accepted role of TF as membrane receptor for FVII, the inventors have shown that TF is also a potent FXa stimulator. TF acts as stimulator of FXa producing a significant enhance in its proteolytic activity. “Stimulators of FXa” as used in this description makes reference to all forms of FXa, such as FXa soluble and FXa bound to prothrombinase complex.
It is well known that FXa at picomolar concentrations is unable to produce any effect on coagulation, even in the presence of its well known cofactor, FVa (see table 9). Therefore, under these conditions prothrombinase complex is not active. Surprisingly, in the presence of TF (i.e. injury or exogenous administration), FXa at picomolar concentrations (i.e. physiological basal concentrations or exogenously administered) cause prothrombin hydrolysis, leading to fibrin clot formation, even in the absence of FVII/FVIIa (table 7).
In the present patent application the inventors describe that TF::FXa interaction is a new trigger coagulation mechanism independent of the TF::FVIIa complexes and the extrinsic coagulation pathway.
Finally, it is well known that there are certain platelet diseases occurring with disorders in platelet aggregation and a greater tendency of hemorrhagic episodes, amongst which Glanzmann's disease and the Bernard-Soulier Syndrome stand out, in which congenital defects affecting the fibrinogen receptor or the Gp1b receptor, respectively, have been disclosed. On the other hand, severe hemorrhagic episodes are present in congenital and acquired thrombocytopenic disorders when platelet count decreases below 20,000 per μl.
In the present patent application, the inventors have demonstrated that lipidated TF is also effective in the treatment of hemorrhages present in congenital, acquired platelet diseases and severe Thrombocytopenic disorders (below 9,000 per μl).
2. Coagulation Pathology
Congenital deficiencies of each coagulation factor can be associated with the occurrence of hemorrhages and generally involve a single protein; thus, for example, hemophilia A is a hereditary hemorrhagic disease affecting FVIII. Acquired coagulation diseases occur in individuals with no prior history of bleeding and may have multiple sources; by way of illustration, the presence of inhibitors specific for coagulation factors may occur in individuals who have been subjected to many transfusions. Although acquired coagulation factor deficiencies are an unknown etiological entity also causing severe hemostasic problems, they are also one of the most important problems in multiple transfusions to which patients with congenital coagulopathies are subjected. Other important source of acquired coagulation disorders are anticoagulant therapies, such as heparin and warfarin drugs. A significant percentage (5-10%) of patients treated with anticoagulant drugs present bleeding episodes most of them are difficult to manage.
As it has been previously mentioned, congenital and acquired platelet disorders can be also associated with hemorrhages. Platelet count decreases (below 20.000 per μl) may cause fibrin clot impairment frequently accompanied with severe bleeding episodes.
The currently available therapeutic arsenal in a mild/moderate or severe/fatal hemorrhage (by surgery or external trauma) is very limited. There are different hemostatic agents that are able to accelerate blood coagulation and preventing hemorrhages, for example (1) human-derived blood products, such as coagulation factor concentrates and local hemostatic agents, such as fibrillar collagen, fibrin glue and prothombin complex concentrates; (2) human recombinant proteins; (3) antifibrinolytics drugs, such as aminocaproic acid, tranexamic acid; and (4) inorganic local hemostatic agents, such as silica and caolin surfaces.
The following critical limitations have been reported:
(1) Intravenous administration
(2) Special device requirement for administration
(3) Narrow therapeutic focus
(4) Inappropriate or troublesome treatment to be administrated in specific bleeding episodes
(5) Lack of acute effect
(6) Instability of fibrin clot
(7) Very dangerous side-effects
(8) Expensive treatment
Human Derived Blood Products (Coagulation Factor and Platelet Concentrates)
It is a high expensive and low available treatment that it must always be intravenously administered. It has a very narrow therapeutic focus, it is only useful to treat their specific deficiency. It is inappropriate to topically treat any kind of bleeding episodes. It is not useful to acutely treat an hemorrhage because it requires long administration protocols to be effective, and above all is a very dangerous treatment: 20% of hemophilic patients have developed hepatitis, 5% HIV, and up to 15% present plasmatic antibodies against FVIII or FIX (acquired hemophilia) that requires special and very expensive substitutive treatments (immunosuppressant, high doses of coagulation factors, plasmapheresis, etc). For these reasons, public health organizations (WHO, FDA, EMEA, etc) are very interested in the development of new hemostatic agents better than coagulation factor concentrates.
Local Hemostatic Agents
It is an expensive treatment inappropriate to be administrated in some bleeding episodes (i.e. epistaxis), troublesome for dental treatment, form unstable fibrin clot, and as coagulation factor concentrates have the same potential dangerous side-effects. They should not be used in patients who have never received human-derived blood products or those who are receiving treatment with recombinant FVIII or FIX because of the potential risks of human viral transmission.
Human Recombinant Proteins
It is the most expensive treatment (average cost of 6,000 ε) only available for developed countries. As coagulation factor concentrates, it must always be intravenously administered, has a very narrow therapeutic focus because it is only useful to treat their specific deficiency, is inappropriate to topically treat hemorrhages, is not useful to acutely treat an hemorrhage because it also requires long administration protocols to be effective, and although no human viral transmission has been reported, the same percentage of acquired hemophilia has been described (up to 15% present antibodies anti FVIII or FIX). As coagulation factor concentrates, public authorities greatly limit its use.
Antifibrinolytic Drugs
They have a narrow therapeutic focus this being its most relevant limitation. These drugs require previous fibrin clot formation to be effective. Therefore, they are only useful in healthy subjects, however when fibrin clot is inappropriately formed (i.e. congenital coagulopathies, such as hemophilia, FVII deficiency) their therapeutic efficacy dramatically decreases. Moreover, they are not useful to acutely treat a hemorrhage because they also require long administration protocols to be effective.
Inorganic Local Hemostatic Agents
The most important restriction for the use of these hemostatic agents is that they are inappropriate to be administrated in much kind of hemorrhages, such as epistaxis, dental, and surgical. Moreover, a painful exothermic reaction has been reported, reducing significantly its use only for mucocutaneous bleeding in critical situations (wars).
In conclusion, surprisingly there are no drugs available today useful for the topical treatment of a simple episode of epistaxis or gingival dental bleeding after brushing one's teeth or simply due to an everyday wound caused by shaving, due to the punctured vein in a blood extraction, or due to the wound from an accidental fall in the street. The problem is further aggravated in the case of patients with hemorrhagic diathesis, for example with congenital coagulopathies of the hemophilia type or the von Willebrand disease or patients with congenital platelet disorders, of the Glanzmann's disease type or the Bernard-Soulier syndrome, or acquired coagulopathies. These patients have serious problems with day to day living and in a simple dental extraction or in any minor trauma causing a bleeding wound they have no medical treatment available to improve their quality of life. The problem obviously becomes greater when these patients suffer an external trauma or severe bleeding accident since their life is at serious risk. In all these situations, the only available pharmacological tool is the administration of human plasma containing the deficient factors or the human recombinant factor specific for each coagulation factor. All these therapies imply using the parenteral route and, therefore, are not designed to be used with great frequency, as would be the case, for example, in any daily mild or moderate bleeding. Finally, it is widely accepted that new local hemostatic agents without the limitations previously described, will represent a significant improve of present treatment which will reduce both the cost and the high prevalence of side-effects.
3. Background of the Invention
Until now it has been accepted that TF is the main element responsible to trigger blood coagulation. For coagulation to begin, it is absolutely necessary activation of FX to FXa to start prothrombin hydrolysis. The source of this FXa has mainly been attributed to the interaction of FVIIa with its receptor, TF. Although it has been described that FXa is present in platelet granules and that it may be exposed on the surface when its activation takes place, the physiological concentrations of FXa (<150 pM) present in blood are insufficient to begin thrombin formation, even in the presence of its cofactor, FVa and of a platelet procoagulant surface (FIG. 1). Therefore, it is currently accepted that coagulation can only start when FXa basal concentrations significantly increase. The source of the increase of FXa basal concentrations has always been attributed to both, the TF::FVIIa complex and FIXa proteolytic activities (FIG. 2).
Lipidated TF recombinant proteins have been able to accelerate only in vitro conditions, coagulation in both healthy and hemophilic blood samples, attributing this action to the classic role assigned to TF as FVIIa receptor. However, in the same experimental conditions, non-lipidated TF has demonstrated a complete lack of effect, which indicates that lipidization is necessary to achieve TF functionality (section 6.1 of the results).
The use of lipidated TF as a topical hemostatic agent has never been described as a single treatment for mild, severe and lethal bleedings (traumatic or surgical arterial and venous hemorrhages).
European patent EP 266993 discloses the use of non-lipidated TF as a hemostatic agent for parenteral treatment of hemorrhagic syndromes. However, the same patent discloses the important differences in activity between the non-lipidated TF claimed by EP 266993 and the lipidated TF object of the present patent application.
In fact, it is well known that lipidated TF is active in vitro conditions and their parenteral administration immediately initiates disseminated intravascular coagulation with fatal consequences. In contrast, non-lipidated TF is not active in vitro conditions, however it has been claimed (EP 266993) for treatment of coagulopathies by parenteral administration.
To date, there are not data about the effect of both, lipidated and non-lipidated TF for single topical treatment of bleeding episodes. In the present patent application, using the tail rat transection model, the inventors have demonstrated that non-lipidated TF was unable to stop bleeding. In contrast, inventors have shown for the first time, that lipidated TF is a useful hemostatic agent to treat topically all kind of hemorrhages, including in pathological (animals treated with heparin and warfarin) and in healthy conditions (control rats without alteration of coagulation). Overall, indicates that lipidated and non-lipidated TF are clearly different compounds. Consequently, the use of lipidated TF as an agent for the single topical treatment of hemorrhages is not obvious for a person skilled in the art.
On the other hand, EP 266993 was performed according to the state of the art which postulates that the serine-protease FVII is only activated when bound to its receptor, TF. Therefore, according to EP 266993 and to the state of the art, when FVII/FVIIa is not present, TF must not be active. It was unobvious to any person skilled in the art to think of TF (lipidated and non-lipidated) could be effective in the treatment of FVII deficient patients. Inventors have discovered that even in the absence of FVIIa, TF acts as a cofactor for FXa. This finding is fundamental to understand that non-lipidated TF can also act as parenteral hemostatic agent for hemorrhage in defective FVII patients.
International patent application WO 94/02172 teaches that the temporarily inhibition of one or more natural anticoagulants by systemic administration of an inhibitor of a natural anticoagulant (an antibody) can inhibit microvascular bleeding. Optionally, the inhibitor can be administered in combination with a topical administration of thrombin or lipidated TF. It is important to point out that the use of both compounds was always as optional adjuvant treatments and never as single treatment. Moreover, WO 94/02172 claims the use of these synergistic treatments only for capillary bleeding (microvascular bleeding, i.e. burns, inflamed visceral surfaces, bleeding liver surfaces . . . ), and never for severe or lethal hemorrhages caused by surgery or external trauma involving arterial and venous injury. However, the inventors of WO 94/02172 admit that, although they observed a synergistic effect when topically administering thrombin in combination with the systemic treatment, no such synergistic effect was observed when administering topically TF (WO 94/02172, FIG. 3 and page 22, lines 14-15). Moreover, the dose claimed in WO 94/02172 ranges very high, from 0.1 to 10 mg.
Contrary to WO 94/02172, the present patent application is claiming the single treatment of mild/moderate to severe/lethal arterial or venous hemorrhage with lipidated TF alone showing examples in which such treatment is effective at a 1.2 μ/ml of active protein dose for traumatic hemorrhages (see severe model in tables 24 and 25). Such a surprising and extraordinarily effective treatment has not been described until now, because it was unobvious for any person skilled in the art that lipidated TF acts as a stimulator of FXa proteolytic activity. Inventors have discovered that in the absence of FVIIa, TF acts as a cofactor for FXa, even in the absence of its well known cofactor, FVa. This finding is fundamental to understand that lipidated TF alone can act as a hemostatic agent for severe hemorrhage in healthy and pathological conditions.
U.S. Pat. No. 4,721,618 teaches that the intravenously administered synergistic mixture of phospholipids (PCPS) and FXa at high concentrations (0.2 to 0.5 U/Kg) may bypass the Factor VIII: C deficiency in a hemophilic mammal, so that the cascade process of blood clotting may continue. The suggested high concentrations of FXa are active without the need of PCPS lipid vesicles (which can enhance this activity). Also, WO 02/086118 teaches that compositions that include a mixture of at least one specific phospholipid and at least one serine protease-activated blood coagulation factor are useful for treating blood coagulation disorders decreasing the need for administered blood coagulation factors.
Furthermore, ellagic acid and other accelerators such as zeolite, silica and inorganic oxide materials have been used to enhance blood coagulation and claimed in WO 02/30479.
According to WO 02/086118, a coagulation factor is defined as a serine protease-activated blood coagulation factor (page 5, lines 13-15). Therefore, TF may not be considered as a blood coagulation factor, because TF is not a serine protease, but a specific cell surface receptor for factor VIIa.
In the present patent application the inventors have demonstrated that phospholipids (phosphatidylserine and phosphatidylcholine at different percentages and molarities) do not increase the procoagulant effect mediated by lipidated TF (table 21), which indicates that the synergistic effect claimed by U.S. Pat. No. 4,721,618 and WO 02/086118 is exclusive for serine protease-activated blood coagulation factors, but not for membrane receptors such as TF. Surprisingly, when lipidated TF was simultaneously combined with negatively charged inorganic surfaces (NCIS) a significant synergistic effect was observed in vitro and in vivo experimental conditions. NCIS used in this description are constituted by a mixture of lipids and a blood coagulation accelerator, ellagic acid. The lipids have net negative charge, what means that the lipidic mixture can include neutral or zwiterionic lipids, but it must contain a certain amount of negatively charged lipids that confers anionic character to the mixture. By way of an illustrative, non limiting example, negatively charged lipids can be sphingolipids (such as ceramide-1-phosphates, glicosilated phosphatidylethanolarnine, hydroxylated or non hydroxylated sulfatides, gangliosides) and glycerol-based lipids (such as phosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphates, phosphatidic acids, phosphatidylglicerols, cardiolipins). There are commercially available NCIS such as Dade® Actin® (Dade Behring) trademark, e.g., Dade® Actin® FS.
In conclusion, the present patent application describes a new local hemostatic agent characterized by the following advantages respect to commercially available drugs:                (1) Easy topical administration        (2) No special device requirements for its topical administration        (3) Broad therapeutic focus (deficits of FV, FVII, FVIII, FIX, FX, FXI, FXII, and FXIII)        (4) Appropriate treatment to be administrated in all kind of bleeding episodes (epistaxis, dental bleeding, mucocutaneous, traumatic and surgery hemorrhages)        (5) Exerts a potent acute effect        (6) Physiological fibrin clot formation (extreme clot stability)        (7) Without side-effects        (8) Low cost treatment        
The main goal of WHO, FDA, EMEA and other public health authorities is to reduce the dangerous side-effects associated to the consumption of human-derived blood products blood. The present invention has been designed to cover these unmeet needs.